All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Immune therapy involves the exposure of components of the immune system to various elements (cytokines, disease associated antigens and natural metabolites) to combat disease processes in which a dysregulated immune response is thought to play a role. Immune dysregulation is thought to play a major part in the pathogenesis or disease course of a great number of disease processes, including various neoplastic, inflammatory, infectious and genetic entities.
These disorders can be perceived as a dysbalance between pro-inflammatory (Th1) and anti-inflammatory (Th2) cytokines, and few of them are described herein below.
The role of the immune system in the pathogenesis of inflammatory bowel disease Inflammatory bowel diseases (IBD) are common gastrointestinal disorders, that can be perceived as being the result of a dysbalance between Th1-pro-inflammatory, and Th2-anti-inflammatory subtypes of immune responses.
There are several extra-intestinal manifestations that accompany IBD, for example: autoimmune phenomena; immune complexes have a role in target organ damage; and, immunosuppressive agents such as glucocorticoids, azathioprine, methotrexate and cyclosporin are used to alleviate the disease. Patients with IBD have antibodies against components of colon cells and several different bacterial antigens. These antigens gain access to the immune system as a consequence of epithelial damage. Abnormalities of T cell-mediated immunity, including coetaneous anergy and diminished responsiveness to T cell stimuli, have also been described in these patients. In addition, changes in mucosal cell mediated immunity were identified, including increased concentrations of mucosal IgG cells and changes in T cells subsets, suggesting antigen stimulation. Exposure of target antigens after infectious, immune, or toxic damage, leads to activation of mucosal immune cells resulting in cytokines that lead to mucosal inflammatory response. Secretion of pro-inflammatory cytokines such as IFNγ, contributes to an increase in mucosal permeability, and has been described in animal models of IBD. Similarly, an increase in collagen synthesis mediated by IL1 and IL6 can be detected in these animals. A Th1-mediated granulomatous colitis model has been established by the adoptive transfer of normal CD45RB T cells from Balb/C mice into CB-17 scid mice. CD4 cells from CD45RB were shown to prevent the disease when injected together with the CD45RB population. This prevention could be reversed by adding antibodies to TGFβ1.
The Th1/Th2 Dysbalance in Inflammatory Bowel Disease
Both CD4 and CD8 lymphocytes can be typed as either Th1 cells that produce IL-2 and IFNγ, or Th2 cells that produce IL-4, and IL-10. The way the immune system responds to foreign and self antigens, is the result of a balance between the two subtypes of responses. A Th1 type response is involved in the pathogenesis of several autoimmune and chronic inflammatory disorders such as IBD. Thus experimental colitis and IBD in humans can be perceived as a dysbalance between pro-inflammatory Th1-type and anti-inflammatory Th2-type cytokines. It has been recently shown, in both animals and humans, that anti-inflammatory cytokines such as IL-10 can downregulate the pro-inflammatory effects of Th1-mediated cytokines, thereby alleviating immune-mediated disorders.
The Role of the Immune System in the Pathogenesis of Non-Alcoholic Steatohepatitis
Non-alcoholic steatohepatitis (NASH) is a clinico-pathological entity consisting of hepatic fat accumulation, inflammation and fibrosis in patients who have no history of alcohol consumption. It may progress to cirrhosis in 20% of cases and is considered the most common cause of cryptogenic cirrhosis in the Western world. NASH is common in patients who suffer of other metabolic disturbances, which are suggested to play a contributing role in the pathogenesis of the disorder. These include insulin resistance, obesity-related ATP depletion, increased free-fatty-acid beta peroxidation, iron accumulation, antioxidant depletion, and leptin deficiency. Yet no therapeutic intervention, including weight loss, tight diabetic control, normalization of lipid levels and antioxidant treatment have consistently shown an alteration in the natural progression of the disorder.
Most information about NASH has been derived from two mammalian models: leptin-deficient ob/ob mice and leptin-receptor deficient fa/fa Zucker rats. Leptin is a protein that is involved with the regulation of body weight. Its deficiency in rodents and humans results in a severe form of ‘metabolic syndrome’ (formerly termed syndrome X) consisting of morbid obesity, glucose intolerance, hyperlipidemia, and severe hepatic steatosis. Yet, as mentioned above, no intervention aimed at correcting some of these metabolic disturbances have resulted in an amelioration of the hepatic steatosis, fibrosis, and inflammation.
Recent evidence suggests that the immune system may play a pivotal role in the pathogenesis of NASH in the leptin deficient models. In leptin deficient mice, defective hepatic macrophage (Kupffer cell) response has been observed after liver injury induction by lipopolysaccharide. In similar models, LPS induction of IL6 was greatly enhanced, while that of IL10 was inhibited. Ob/ob mice hepatic macrophages were observed to produce more IL12 and less IL15 than control mice in response to LPS challenge, which may explain the significant reduction in the number and function of NKT lymphocytes observed in these mice. Other observations have shown a reduction in the number of CD4 T lymphocytes in the blood and liver of leptin-deficient ob/ob mice. This may explain the relative resistance of leptin-deficient mice to Concanavalin A hepatitis, which is mediated by CD4 T lymphocytes.
The Th1/Th2 Dysbalance in Non-Alcoholic Steatohepatitis
CD4 and CD8 lymphocytes are classified as either Th1 cells that produce IL-2 and IFNγ, or Th2 cells that produce IL-4 and IL-10. The immune system responds to foreign and self-antigens by a shift in balance between the two subtypes of responses [Weiner, H. L. et al., Immunol. Today 18: 335-343 (1997)]. Usually the Th1 type response causes a pro-inflammatory reaction, while anti-inflammatory cytokines such as IL10 shift the balance towards an anti-inflammatory Th2 reaction, thereby alleviating immune-mediated disorders. NKT cells, in response to different endogenous and exogenous stimuli, are believed to play a major role in the direction of the immune system towards either the Th1 or Th2 pathways.
Leptin has been shown to play a major role in the immune regulation of the balance between Th1 and Th2 response. In the leptin-deficient ob/ob mice NASH model an alteration of the number and function of NKT cells has been suggested to tilt the immune system towards the Th1 response. This is suggested to result in an increased sensitivity to LPS induced hepatotoxicity and a unique resistance to the hepatotoxic effects of Concanavalin A. The difference may be in their different pathogenic mechanisms. The former depends upon the action of the innate hepatic immune system, which is hyperactive in the leptin-deficient mice, while the latter is dependent upon the activation of NKT-lymphocytes, which are suppressed and defective in the leptin deficient mice.
The Immune System and Obesity
The immune system and the regulation of adipose tissue metabolism appear to be closely interlinked. Up to fifty percent of cells within adipose tissues are composed of non-adipose cells, including many immunocytes. Most research has been focused on the immunological consequences of morbid obesity. Immunological alterations which are known to exist in obese animals and humans include reduced DTH and mitogen-stimulated lymphocyte proliferation responses, impaired phagocyte number and function, attenuation of insulin induced lymphocyte cytotoxicity, and changes in the CD4/CD8 ratio, especially during weight loss attempts.
Adipose cells are known to secrete pro-inflammatory cytokines including TNF-β [Hotamisligil, G. S. et al., Science 259:87-91 (1993)] and IL6, which are both related to the level of adiposity. Some of these cytokines are considered to have metabolic effects such as insulin resistance mediated by TNF-β and lipoprotein lipase inhibition mediated by IL6. TNF-βknockout mice have higher insulin sensitivity and improved lipid profile than their normal littermates. Other components of the immune system, which are produced by adipose cells, include the protein adipsin, which is an integral part of the alternative complement system, and functions identically to human complement factor D.
Little information is known about the role of the immune system as a mediator of obesity, but several recent studies suggest that the immune system may have an important contributory role in the development of obesity. Several cytokines are known to act as adipose tissue regulators. TNF-β suppresses the expression of β3 adreno-receptors on adipose cells, which are involved in sympathetically mediated lipolysis, while IL1 stimulates adipose leptin secretion. The metabolic activity rate of adipose cells has been observed to be closely correlated to their distance from the closest lymph node, through a mechanism which is partly mediated by IL4, IL6 and TNF-β.
These observations, which point to the fact that obese animals and humans may also be suffering of various alterations in the different arms of the immune system, suggest that modulation of the immune system may change some of the pathogenic mechanisms responsible for the development of morbid obesity.
Neurodegenerative disorders, which are chronic and progressive, are characterized by selective and symmetric loss of neurons in motor, sensory, or cognitive systems. Delineation of the patterns of cell loss and the identification of disease-specific cellular markers have aided in nosologic classification, senile amyloid plaques (SP), neurofibrillary tangles (NFT), neuronal loss, and acetylcholine deficiency define Alzheimer's disease (AD), Lewy bodies and depletion of dopamine characterize Parkinson's disease, cellular inclusions and swollen motor axons are found in amyotrophic lateral sclerosis, and γ-aminobutyric acid-containing neurons of the neostriatum are lost in Huntington's disease.
It is well accepted that the intrinsic differences in cellular metabolism of distinctive brain regions and neuronal populations underlie the selective cellular response to various environmental neurotoxicants, and neuropathological conditions, therefore causing distinct neurodegenerative diseases.
Amyloid diseases are caused by the misfolding of proteins into structures that lead them to cluster together, forming microscopic fibril or plaques, which deposit in internal organs and interfere with normal function, sometimes lethally.
These diseases include Alzheimer's disease, Parkinson's disease, and the peripheral nervous system disease familial amyloid polyneuropathy (FAP). In Alzheimer's disease, these clumps are termed amyloid plaques and consist primarily of the amyloid-beta (Aβ) peptide. In the case of Alzheimer's disease, these fibrils cause degeneration of nerve cells in areas of the brain that are crucial for memory. The Aβ peptides possess neurotoxic properties also in their soluble form. In Parkinson disease, they are called Lewy bodies and contain the protein α-synuclein. FAP, a collection of more than 80 rare amyloid diseases are caused by the misfolding of the protein transthyretin (TTR), which the liver secretes into the bloodstream to carry thyroid hormone and vitamin A.
In the FAP diseases, mutations in the TTR protein are known to play a direct role in causing the disease. These changes alter protein folding in such a way as to predispose the proteins to misfold and accumulate into microscopic fibrils, which can grow into protein plaques.
In Alzheimer's disease, the cause of misfolding is not so obvious. A number of mutations are associated with rare forms of familial Alzheimer's disease, but not with most common cases (about 95 percent of the cases). This suggests there must be a more common cause of Alzheimer's disease.
Traumatic head injuries are a major risk factor for later developing Alzheimer's disease. The body responds to such injuries with inflammatory reactions that cause the release of components of lipid membranes, such as cholesterol. Inflammation can lead to the production of reactive oxygen species such as ozone, which can trigger pathological changes in other molecules in the body, like cholesterol.
Alzheimer's disease (AD) is a progressive neurodegenerative incurable disease. It is the major cause of dementia in the elderly. The estimated number of patients is approximately 20 million worldwide and is expected to keep growing as the world population ages. In the USA, an estimated 10% of Americans over the age of 65 and half of these over 85 have AD.
The onset of the disease is characterized by impaired memory but with disease progression other intellectual skills decline. Later, erratic behavior, delusions and a loss of control over body functions occur. The major brain pathological features include the senile amyloid plaques (SP), composed of Aβ peptide, and the neurofibrillary tangles (NFT), which are aggregations of the hyperphosphorylated microtubular protein tau.
Cholinergic dysfunction as well as oxidative stress are implicated in the disease pathogenesis. As these cellular changes progress, neurons are lost in the hippocampus, entorhinal cortex, and association areas of the neocortex [reviewed in Mayeux R. (2003) Annu. Rev. Neurosci. 26:81-104; Selkoe D. (2001) Physiological Rev. 81:741-766].
The etiology of AD is complex and involves a combination of factors including genetic, immune, endocrine and environmental factors. One such AD-related factor that is attracting recently great attention is the role of cholesterol metabolism and trafficking. There is accumulating data in support of the hypothesis that altering in the cholesterol levels influences the development of AD by affecting the formation of Aβ peptide, its distribution within cholesterol rich membranes and its fibrillogenesis.
Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, with a prevalence of two percent among people over the age of 65 years. The disease is mostly sporadic, but familial forms are recognized as well. Parkinson disease (PD) targets dopaminergic neurons in the substantia nigra, resulting in motor disturbances such as resting tremor, bradykinesia, and rigidity. There is a substantial clinical overlap between Alzheimer's disease and Parkinson's disease. Dementia develops in approximately 20 to 30 percent of patients with Parkinson's disease, and the brains of these patients often contain Lewy bodies, SP and NFT.
The third common neurodegenerative diseases are the motor neuron diseases. The most common motor-neuron disorder is amyotrophic lateral sclerosis (ALS), which usually begins in the fifth and sixth decades of life. The illness is usually sporadic, but in 1 to 10 percent of patients it is familial, being inherited as an autosomal dominant trait. In a typical patient, muscles innervated by both brain stem and spinal cord atrophy as lower motor neurons die, although those that control eye movements and bowel and bladder function are spared. The prognosis is grave, with death occurring in three to five years in 95 percent of patients.
Another neurodegenerative disease is the Huntington's disease, which is an autosomal dominant disorder with high penetrance. The characteristic findings of progressive chorea and dementia are caused by severe neuronal loss, initially in the neostriatum and later in the cerebral cortex.
Although the regions and cells that degenerate in these various illnesses and insults are distinct, several features are common to many of these conditions and include aberrant protein interactions and aggregation, mitochondrial dysfunction, altered antioxidant defenses, oxidative stress, inflammation and apoptosis.
Different neurological disorders, known as “taupathies” have been recently described. In these disorders it has been suggested that modifications in the microtubule-associated protein tau could cause neural degeneration in specific regions. Although these regions are different in the different taupathies, some common features appear to occur in all of them: neurofibrillary tangles (NFT), which are aggregations of the abnormal hyperphosphorylated microtubular protein tau.
Abnormal tau proteins are often seen as mechanisms that can lead to brain degeneration in Alzheimer's disease and other neurodegenerative disorders known as taupathies. In all taupathies, there are neuropathologic aggregates of paired helical filaments and/or straight filaments composed of aberrantly phosphorylated tau proteins in central nervous system neurons or glia.
Although impressive advances in understanding of these diseases have been made, still the mechanisms of brain degeneration are not resolved, and no effective drug is available. To date only the secondary degenerative effects have been amenable to therapy.
WO 2005/032462, which is a previous publication by the present inventors, discloses the general use of intermediary metabolites and preferably, glucocerebrosides, in the treatment of immune-related disorders. The inventors have further showed recently that β-lactosyl-ceramide may be used as a preferred β-glycolipid for immune-modulation (IL2006/001217). The inventors further demonstrated a clear synergistic effect of a particular combination of two β-glycolipids, preferably a mixture of β-lactosyl-ceramide (LC) with β-glucosylceramide (GC), which may be used as a powerful medicament for the treatment of immune-related disorders.
Still further, the inventors recently demonstrated the use of β-glycolipids, particularly GC and LC, in the treatment of neurodegenerative disorders and CNS related inflammatory autoimmune disorders.
In another publication, WO03/027058, the present inventors disclosed synthetic sphingolipid derivatives, particularly for use in treating lipid storage diseases.
The present invention now clearly demonstrates the use of synthetic derivatives of β-glycolipids, and particularly of the compounds of Formulas I, II, III and IV, for the treatment of pathologic disorders.
It is therefore an object of the invention to provide novel synthetic derivatives of β-glycolipids, as well as compositions thereof and methods for treating pathologic disorders such as immune-related disorders and neurodegenerative disorders.
These and other objects of the invention will become clearer as the description proceeds.